Polyhydroxyalkanoates for in vivo applications

ABSTRACT

Polyhydroxyalkanoates (PHAs) from which pyrogen has been removed are provided for use in numerous biomedical applications. PHAs which have been chemically modified to enhance physical and/or chemical properties, for targeting or to modify biodegradability or clearance by the reticuloendothelial system (RES), are described. Methods for depyrogenating PHA polymers prepared by bacterial fermentation processes are also provided, wherein pyrogens are removed from the polymers without adversely impacting the polymers&#39; inherent chemical structures and physical properties. PHAs with advantageous processing characteristics, including low melting points and/or solubility in non-toxic solvents, are also described. PHAs are provided which are suitable for use in in vivo applications such as in tissue coatings, stents, sutures, tubing, bone and other prostheses, bone or tissue cements, tissue regeneration devices, wound dressings, drug delivery, and for diagnostic and prophylactic uses. Properties which are selected for include degradability, elasticity, inclusion of functional groups or derivatized groups, which can in turn be used to attach targeting agents, and bioadhesion.

CROSS-REFERENCE TO ELATED APPLICATIONS

[0001] Benefit is claimed of priority to U.S. Ser. No. 60/046,211,entitled “Biocompatible Polyhydroxyalkanoates” filed May 12, 1997 bySimon F. Williams; Ser. No. 60/054,289, entitled “Derivatization of PHAsfor Biomedical Applications” filed Jul. 31, 1997 by David Martin; Ser.No. 60/063,501, entitled “Polyhydroxy Alkanoate Stents” filed Oct. 24,1997 by Simon F. Williams and David P. Martin; and Ser. No. 60/065,921,entitled “Method for Making Biocompatible Polyhydroxyalkanoates” filedNov. 17, 1997, by Simon F. Williams and David P. Martin.

BACKGROUND OF THE INVENTION

[0002] The present application is generally directed topolyhydroxyalkanoates polymers and methods of preparation to removepyrogen and use thereof in a variety of biomedical applications,including tissue engineering, wound dressings, drug delivery, and inprosthetics.

[0003] Polyhydroxy alkanoates (PHAs) are polymers with repeating hydroxyacid monomeric units. PHAs have been reviewed in several publications,including Byrom, “Miscellaneous Biomaterials,” in Biomaterials (D.Byrom, ed.) pp. 333-59 (MacMillan Publishers, London 1991); Hocking andMarchessault, “Biopolyesters” in Chemistry and Technology ofBiodegradable Polymers (G. J. L. Griffin, ed.) pp. 48-96 (Chapman andHall, London 1994); Müller and Seebach, Angew. Chem. Int. Ed. Engl.,32:477-502 (1993); Steinbüchel, “Polyhydroxyalkanoic Acids,” inBiomaterials (D. Byrom, ed.) pp. 123-213 (MacMillan Publishers, London1991); and Williams and Peoples, CHEMTECH, 26:38-44 (1996).

[0004] Polyhydroxybutyrate (PHB) and polyhydroxybutyrate-hydroxyvalerate(PHBV) have been used commercially as a biodegradable replacement forsynthetic commodity resins, and have been extensively studied for use inbiomedical applications. Examples of these biomedical applicationsinclude controlled release (Pouton and Akhtar, Adv. Drug Delivery Rev.,18:133-62 (1996)), tablet formulations, surgical sutures, wounddressings, lubricating powders, blood vessels, tissue scaffolds,surgical implants to join tubular body parts, bone fracture fixationplates, and other orthopedic uses, (Hocking and Marchessault,“Biopolyesters” in Chemistry and Technology of Biodegradable Polymers,(G. J. L. Griffin, ed.) pp. 48-96 (Chapman and Hall, London 1994) andreferences therein); European Patent Application 754 467 A1 (Bowald, etal.). See also Saghir Akhtar, Ph.D. Thesis for the University of Bath,1990, “Physicomechanical Properties of Bacterial P(HB-HV) Polyesters andTheir Uses in Drug Delivery”. PHBV has been used to sustain cell growthand in tissue reconstruction (see, e.g., Rivard, et al., J. Appl.Biomat., 6:65-68 (1995)). However, PHBs and PHBVs have been shown toinduce acute inflammatory responses when implanted in vivo (Akhtar atpp. 50-51, and references cited therein).

[0005] Biodegradable polymers for medical uses must be biocompatible anddegrade into non-toxic metabolites. Medical devices must also benonpyrogenic, i.e., the products must not produce fever reactions whenadministered to patients. The presence of bacterial endotoxin (which isan integral component of the outer cell surface of Gram-negativebacteria), in the product is by far the largest concern of manufacturersin achieving nonpyrogenation. (Weary and Pearson, BioPharm., 1:22-29(1988)). The U.S. Food and Drug Administration (FDA), for example,requires the endotoxin content of medical devices not exceed 20 U.S.Pharmacopeia (USP) endotoxin units (EU) per device, except for thosedevices that contact the cerebrospinal fluid, where the content must notexceed 2.15 USP endotoxin units per device. Acceptable endotoxin levelsmay need to be even lower for some applications, where the polymer is tobe used for particularly sensitive applications. Therefore, indeveloping PHAs for use in medical devices, the materials must meet thespecific requirements set for endotoxin content, particularly for PHAsderived by fermentation of gram-negative bacteria, where the polymersare exposed to large amounts of endotoxin in the cell culture.

[0006] U.S. Pat. No. 5,334,698 to Witholt, et al. discloses sutures,films, skin grafts, and bone grafts prepared from an optically activepolyester isolated from Pseudomonas oleovorans cells. PCT ApplicationPublication WO 96/00263 (Eggink, et al) discloses an aqueous latex-likePHA dispersion, wherein the PHA includes saturated or unsaturated3-hydroxy fatty acids having a carbon chain length of 6-14. Hocking andMarchessault, “Biopolyesters” in Chemistry and Technology ofBiodegradable Polymers, (G. J. L. Griffin, ed.) pp. 48-96 (Chapman andHall, London 1994) also discloses polyesters with functionalizedside-chains prepared by bacteria upon modifying the feed substrate, andtheir use for preparing drug delivery systems. These materials wouldinherently include endotoxin, and there is no disclosure of any methodsfor removing endotoxins or procedures for providing depyrogenatedpolymers suitable for in vivo medical use.

[0007] Despite the large amount of literature describing production,purification, and applications development of PHAs, there are currentlyno reported methods specifically for depyrogenating PHA polymers. PHAshave a relatively high affinity for endotoxins, complicating the use ofroutine procedures for depyrogenation. Thus, there is a need to developmethods for depyrogenating PHA polymers, particularly when they areproduced by fermentation in Gram-negative bacteria.

[0008] Even aside from the issue of pyrogenicity, there remains a needto develop additional biodegradable polymers for in vivo use,particularly polymers with alternative physical and chemical properties.These properties include characteristics relevant to ease of processing,as well as suitablility for the end use. An important physical propertyfor processing of the polymers is the melting point or glass transitiontemperature of the materials. PHB, PHBV (0-24% V), PGA and PLGA, forexample, all melt only at relatively high temperatures, above 136° C.This high temperature can be a disadvantage in fabrication if thepolymers are to be combined in the melt with other heat sensitivecomponents. It would be advantageous to have a class of PHAs which havemelting points or glass transitition temperatures below 136° C. for usein biomedical applications. Further, many PHAs are only soluble inpotentially toxic chlorinated solvents. There is thus a need to developlow melting PHAs that can be melt processed at low temperatures and/orcan be dissolved in non-toxic, generally acceptable solvents. However,there is currently no commercial source for polyhydroxyalkanoatematerials with these properties.

[0009] Other properties such as the thermal and mechanical properties,density and crystallinity, are also of interest. These properties can bemodified by mixing or blending PHAs with other materials, or by changingthe PHA composition. The commercially available PHAs, PHB and PHBV, haveonly limited uses. Other PHAs can be used for very differentapplications. For example, the extension-to-break of PHBV ranges fromabout 8 to 42%, whereas the same property for polyhydroxyoctanoate(PHO), a low melting PHA, is about 380% (Gagnon, et al., Rubber World,207:32-38 (1992)). Similarly, PHBV has a Young's Modulus of between1,000 and 3,500 MPa and a tensile strength of between 20 and 31 MPa, incontrast to PHO which has a Young's Modulus of 8 MPa and a tensilestrength of 9 MPa (Gagnon, et al., Rubber World, 207:32-38 (1992)).These properties and others have lead to PHO being classified as athermoplastic elastomer (Gagnon, et al., Rubber World, 207:32-38(1992)). The covalent crosslinking of unsaturated pendant groups ofseveral polyhydroxyalkanoate thermoplastic elastomers has been reported(Gagnon, et al, Polymer, 35:4358-67 (1994)), although the use of thepolymers for preparing medical devices is not disclosed. It would beuseful to develop biocompatible low melting PHAs containing groups whichcan be covalently modified, or which can be subsequently modified toexpose functional groups which can be derivatized, for use in preparingmedical devices.

[0010] Accordingly, it is an object of this invention to providepolyhydroxyalkanoate polymers having most of pyrogen removed, for use inbiomedical applications.

[0011] It is another object of this invention to provide biocompatiblepolyhydroxyalkanoate polymers with low melting points and/or solubilityin non-toxic, non-halogenated solvents.

[0012] It is another object of this invention to providepolyhydroxyalkanoates having desirable properties for use in a varietyof biomedical applications, such as drug delivery, tissue engineering,medical imaging, and the manufacture of prosthetics, stents, andcoatings.

[0013] It is a further object of this invention to provide methods formaking biomedical devices using polyhydroxyalkanoate polymers.

SUMMARY OF THE INVENTION

[0014] Polyhydroxyalkanoates (PHAs) from which pyrogen has been removedare provided for use in numerous biomedical applications. PHAs whichhave been chemically modified to enhance physical and/or chemicalproperties, for targeting or to modify biodegradability or clearance bythe reticuloendothelial system (RES), are described. Methods fordepyrogenating PHA polymers prepared by bacterial fermentation processesare also provided, wherein pyrogens are removed from the polymerswithout adversely impacting the polymers' inherent chemical structuresand physical properties. PHAs with advantageous processingcharacteristics, including low melting points and/or solubility innon-toxic solvents, are also described. PHAs are provided which aresuitable for use in in vivo applications such as in tissue coatings,stents, sutures, tubing, bone and other prostheses, bone or tissuecements, tissue regeneration devices, wound dressings, drug delivery,and for diagnostic and prophylactic uses. Properties which are selectedfor include degradability, elasticity, inclusion of functional groups orderivatized groups, which can in turn be used to attach targetingagents, and bioadhesion.

DETAILED DESCRIPTION OF THE INVENTION

[0015] Biocompatible polyhydroxyalkanoates (PHAs) are provided whereinpyrogens such as endotoxin, present due to the process by which the PHAsare made, are removed without damage to the polymer composition orstructure. In a preferred embodiment, the polymers have melting pointsor glass transition temperatures less than 136° C., and/or are solublein non-toxic non-halogenated solvents.

[0016] I. Polyhydroxyalkanoates

[0017] Several types of polyhydroxy alkanoates are formed in nature byvarious organisms in response to environmental stress. These PHAs can bebroadly divided into three groups according to the length of theirpendant groups and their respective biosynthetic pathways. Relativelyshort pendant groups are the C₃₋₅ hydroxy acids, whereas relatively longpendant groups are C₆₋₁₄ hydroxy acids.

[0018] There are three major types of naturally occurring PHAs. Thefirst type includes only relatively short hydroxy acid monomeric units.The second type include both relatively short and relatively longhydroxy acid monomeric units. The third type includes only relativelylong hydroxy acid monomeric units. Those with short pendant groups, suchas polyhydroxybutyrate (PHB), a homopolymer of R-3-hydroxybutyric acid(R-3HB) units, are highly crystalline thermoplastic materials (Lemoigneand Roukhelman, Annales des fermentations, 5:527-36 (1925)). PHAscontaining the short R-3HB units randomly polymerized with much longerpendant group hydroxy acid units were first reported in the earlyseventies (Wallen and Rohwedder, Environ. Sci. Technol., 8:576-79(1974)). A number of microorganisms which specifically producecopolymers of R-3HB with these longer pendant group hydroxy acid unitsare also known and belong to this second group (Steinbüchel and Wiese,Appl. Microbiol. Biotechnol., 37:691-97 (1992)). In the early 1980's, aresearch group in The Netherlands identified the third group of PHAs,which contains predominantly longer pendant group hydroxy acids (DeSmet, et al., J. Bacteriol, 154:870-78 (1983)).

[0019] PHAs may constitute up to 90% of the dry cell weight of bacteria,and are found as discrete granules inside the bacterial cells. These PHAgranules accumulate in response to nutrient limitation and serve ascarbon and energy reserve materials. Distinct pathways are used bymicroorganisms to produce each group of these polymers. One of thesepathways leading to the short pendant group polyhydroxyalkanoates(SPGPHAs) involves three enzymes: thiolase, reductase, and PHB synthase(sometimes called polymerase). Using this pathway, the homopolymer PHBis synthesized by condensation of two molecules of acetyl-Coenzyme A togive acetoacetyl-Coenzyme A, followed by reduction of this intermediateto R-3-hydroxybutyryl-Coenzyme A, and subsequent polymerization. Thelast enzyme in this pathway, namely the synthase, has a substratespecificity that can accommodate C₃₋₅ monomeric units, includingR-4-hydroxy acid and R-5-hydroxy acid units. This biosynthetic pathwayis found, for example, in the bacteria Zoogloea ramigera and Alcaligeneseutrophus.

[0020] The biosynthetic pathway which is used to make the third group ofPHAs, long pendant group polyhydroxyalkanoates (LPGPHAs), is stillpartly unknown. However, it is currently thought that the monomerichydroxyacyl units leading to the LPGPHAs are derived by the α-oxidationof fatty acids and the fatty acid pathway. The R-3-hydroxyacyl-Coenzymesubstrates resulting from these routes are then polymerized by PHAsynthases (sometimes called polymerases) that have substratespecificities favoring the larger monomeric units in the C₆₋₁₄ range.LPGPHAs are produced, for example, by Pseudomonads.

[0021] The second group of PHAs containing both short R-3HB units andlonger pendant group monomers are believed to utilize both the pathwaysto provide the hydroxy acid monomers. The latter are then polymerized byPHA synthases able to accept these units.

[0022] Roughly 100 different types of PHAs have been produced byfermentation methods so far (Steinbüchel and Valentin, FEMS Microbiol.,Lett., 128:219-28 (1995)). A number of these PHAs contain functionalizedpendant groups such as esters, double bonds, alkoxy, aromatic, halogens,and hydroxy groups. Transgenic systems for producing PHAs in bothmicroorganism and plants, as well as enzymatic methods for PHAsynthesis, are reviewed by Williams and Peoples, CHEMTECH, 26:38-44(1996).

[0023] Two PHAs belonging to the first group, polyhydroxybutyrate (PHB)and polyhydroxybutyrate-co-valerate (PHBV), have been extensivelystudied. PHBV is a copolymer of R-3HB units with 5-24%R-3-hydroxyvaleric acid (R-3HV), and is known commercially as Biopol™(supplied by ICI/Zeneca). These polymers are natural thermoplasticswhich can be processed using conventional polymer technology and whichhave industrially useful properties, such as biodegradability in soiland marine environments and good barrier properties. They arecharacterized by melting points which range from 130 to 180° C., andextensions-to-break of 8 to 42% (see Zeneca Promotional Literature,Billingham, UK 1993).

[0024]1. Polymer Formulas

[0025] The PHAs as described herein can be in the form of homopolymers,block copolymers, or random copolymers. Biocompatible polymers aregenerally defined as those polymers that result in minimal tissuereaction when implanted in vascularized tissue. As used herein,biocompatible polymers are those which do not elicit an acuteinflammatory response when implanted into the muscle of an animal suchas a mouse.

[0026] In some embodiments, the polymers can also be characterized ashaving low endotoxin levels. Preferably, the PHAs are highly purematerials, with purities exceeding 95%, more preferably exceeding 98%.The PHAs may be purified by extraction with or precipitation fromaqueous solutions, organic solvents, supercritical fluids, orcombinations thereof.

[0027] The molecular weight of the polymers is preferably between 300and 10⁷, and, more preferably, between 10,000 and 10,000,000 Daltons.The PHAs preferably contain one or more units, more preferably between10 and 100,000 and most preferably between 100 and 30,000 units of thefollowing formula:

—OCR¹R²(CR³R⁴)_(n)CO—

FORMULA I

[0028] wherein n is an integer, for example, between one and 15,preferably between one and four; and

[0029] wherein R¹, R², R³, and R⁴ are independently selected fromhydrogen, methyl, C₂₋₁₅ straight, branched or cyclic alkyl, alkenyl oralkynyl groups, alkaryl groups, aralkyl groups, heteroalkyl groups,heteroaryl groups, hydroxy groups, thiol groups, disulfides, ethergroups, thiolether groups, ester groups, carboxylic acid groups, aminegroups, amide groups, halogens, nitrogen-substituted radicals; and/oroxygen-substituted radicals.

[0030] Suitable monomeric units include hydroxybutyrate,hydroxyvalerate, hydroxyhexanoate, hydroxyheptanoate, hydroxyoctanoate,hydroxynonanoate, hydroxydecanoate, hydroxyundecanoate, andhydroxydodecanoate units. PHAs including monomers and polymers andderivatives of 3-hydroxyacids, 4-hydroxyacids and 5-hydroxyacids can beused. Representative PHAs are described in Steinbüchel, A. and Valentin,H. E., FEMS Microbiol., Lett., 128:219-28 (1995).

[0031] Preferred PHAs have melting points or glass transitiontemperatures less than 136° C. These materials are referred to as “lowmelting PHAs”. The low melting PHAs specifically exclude thehomopolymer, polyhydroxybutyrate (PHB), and the commercial copolymers ofR-3-hydroxybutyric acid and R-3-hydroxyvaleric acid (PHBV) with avalerate content in the copolymer of between 0 and 24%.

[0032] Although described herein primarily with reference to thepolyhydroxyalkanoate polymers, it is understood that these polymers maybe blended with other polymers, and/or co-polymerized with monomers orother polymers to form polyhydroxyalkanoate copolymers. Examples ofother polymers particularly suited for biomedical applications includebiodegradable polymers such as polyhydroxy acids prepared frompolylactic acid, polyglycolic acid, and copolymers thereof,polycarbonates, polyorthoesters, polyanhydrides, polyphosphazenes,polyamino acids, proteins, and polysaccharides. The term“polyhydroxyalkanoate” refers to polyhydroxyalkanoate polymers, blends,and copolymers, unless otherwise stated.

[0033] 2. Preparation of PHAs

[0034] The PHAs can be prepared from a biological source such as amicroorganism which naturally produces the PHAs or which can be inducedto produced the PHAs by manipulation of culture conditions andfeedstocks, or microorganisms or a higher organism such as a plant,which has been genetically engineered so that it produces PHAs.

[0035] Methods which can be used for producing PHA polymers frommicroorganisms which naturally produce polyhydroxyalkanoates aredescribed in U.S. Pat. No. 4,910,145 to Holmes, et al.; Byrom, D.,“Miscellaneous Biomaterials,” in D. Byrom, Ed., “Biomaterials” MacMillanPublishers, London, 1991, pp. 333-59; Hocking, P. J. and Marchessault,R. H. “Biopolyesters”, G. J. L. Griffin, Ed., “Chemistry and Technologyof Biodegradable Polymers,” Chapman and Hall, London, 1994, pp. 48-96;Holmes, P. A., “Biologically Produced (R)-3-hydroxyalkanoate Polymersand Copolymers,” in D. C. Bassett Ed., “Developments in CrystallinePolymers,” Elsevier, London, Vol. 2, 1988, pp. 1-65; Lafferty et al.,“Microbial Production of Poly-b-hydroxybutyric acid,” H. J. Rehm and G.Reed, Eds., “Biotechnology”, Verlagsgesellschaft, Weinheim, vol. 66,1988, pp. 135-76; Müller and Seebach, Angew. Chem. Int. Ed. Engl.32:477-502 (1993).

[0036] Methods for producing PHAs in natural or genetically engineeredorganisms are described by Steinbüchel, A. “Polyhydroxyalkanoic Acids,”in D. Byrom Ed., “Biomaterials”, MacMillan Publishers, London, 1991, pp.123-213; Williams and Peoples, CHEMTECH, 26:38-44, (1996); Steinbücheland Wiese, Appl. Microbiol. Biotechnol, 37:691-97 (1992); U.S. Pat. Nos.5,245,023; 5,250,430; 5,480,794; 5,512,669; 5,534,432 to Peoples andSinskey; Agostini, D. E. et al., Polym. Sci., Part A-1, 9:2775-87(1971); Gross, R. A. et al., Macromolecules, 21:2657-68 (1988); Dubois,P. I. et al., Macromolecules, 26:4407 -12 (1993); Le Borgne, A. andSpassky, N., Polymer, 30:2312-19 (1989); Tanahashi, N. and Doi, Y.,Macromolecules, 24:5732-33 (1991); Hori, Y. M. et al., Macromolecules,26:4388-90 (1993); Kemnitzer, J. E. et al., Macromolecules, 26:1221-29(1993); Hori, Y. M. et al., Macromolecules, 26:5533-34 (1993); Hocking,P. J. and Marchessault, R. H., Polym. Bull., 30:163-70 (1993); Xie, W.et al., Macromolecules, 30:6997-98 (1997), and U.S. Pat. No. 5,563,239to Hubbs, et al, the teachings of which are incorporated herein.

[0037] PHAs prepared using methods other than bacterial systemstypically do not contain pyrogens, and, accordingly, need not bedepyrogenated. Pyrogens present in bacterial fermentation systems areusually endotoxins, although other toxins may also be present,particularly in Gram positive bacterial systems, and may also be presentin alternative production systems.

[0038] PHAs can also be prepared using chemical syntheses, for example,via the ring-opening polymerization of β-lactone monomers using variouscatalysts or initiators such as aluminoxanes, distannoxanes, oralkoxy-zinc and alkoxy-aluminum compounds (see Ago stini, et al., Polym.Sci., Part A-1, 9:2775-87 (1971); Gross, et al., Macromolecules,21:2657-68 (1988); and Dubois, et al., Macromolecules, 26:4407-12(1993)); or via condensation polymerization of esters (see, for example,U.S. Pat. No. 5,563,239 to Hubbs, et al., and references therein).Researchers also have developed chemo-enzymatic methods to prepare PHAs.For example, Xie, et al., Macromolecules, 30:6997-98 (1997) reported aring opening polymerization of beta-butyrolactone by thermophiliclipases to yield PHB.

[0039] 3. Modification of PHAs

[0040] The PHA polymers can contain or be modified to include othermolecules, such as bioactive and detectable compounds, surface activeagents, other degradable or non-degradable polymers, and materials usedto modify the mechanical properties of the PHAs, such as plasticizers,fillers, and binders. The modifications can involve covalent and/ornon-covalent attachment of molecules to the PHAs. The modifications mayalso include chemical or physical treatment of the PHAs, which maysubsequently be followed by covalent or non-covalent attachment ofmolecules. Covalent modification of PHAs can enhance their usefulnessfor biomedical applications. Newly introduced functional groups may thenserve as sites of covalent attachment, for example, for drugs, cellattachment peptides, and growth factors.

[0041] Functionalized PHAs are polyhydroxyalkanoates containing reactivefunctional groups. Reactive functional groups, such as carboxylic andamino groups, impart new properties to the polymer and provide sites forits covalent derivatization. These groups can be introduced into PHAs ina number of ways. For example, functionalized (or functionalizable)monomers may be incorporated into the PHAs during production of thepolymer. Controlled fermentation conditions have been used to producePHAs with a variety of functional groups in the pendant side chain suchas alkenes, halogens, esters and branched alkyl groups. After isolation,chemical treatments can subsequently convert these functional groupsinto a variety of others, such as carboxylic, amino, and carbonylgroups. Tillman recently has prepared gram quantities of bromo- andalkene-functionalized PHAs.

[0042] Another approach to functionalizing PHAs is to chemically orphysically modify the isolated parent (functionalized) polymer.Modification of the PHA after production, purification, and isolation isparticularly attractive for a number of reasons. Fermentation systemsfor the production of unfunctionalized PHAs are typically the easiest,cheapest, and highest yielding, since this obviates the need forexpensive functionalized monomers. Purification of the unfunctionalizedpolymer is not complicated by the presence of functional groups. Thedegree of polymer modification can be controlled in a derivatizationprocedure after the polymer is isolated and purified.

[0043] The polyester backbone of a PHA is a potential site formodification through aminolysis or transesterification reactions. Thesereactions can be performed on the bulk polymer, or directed selectivelyto the surface of a PHA article. Surface modification has thesignificant advantage in that only the surface of the material ismodified, while bulk modification of the polymer results in a uniformlymodified material. Modifications that cleave the polyester backbone areexpected to cause random chain scission, and, depending upon the levelof polymer modification, should result in a significant reduction of thepolymer MW. As an example of this approach, PHO films have been modifiedwith bioactive molecules, such as biotin, to produce surfaces which bindstreptavadin-HRP conjugates (see Example 17 which follows).

[0044] The pendant side group is also a potential site for modificationof PHAs. Chemical or physical treatments that generate reactive species,such as free radicals, will modify the pendant side chain. While attackof the polymer backbone may also occur, conditions for the selectivemodification of the pendant side chain should be achievable. Gas plasmatreatment is an example of this type of modification. Depending on thetype of gas used to generate the plasma, this type of treatment canintroduce a variety of new functional groups.

[0045] Gas plasma is an ionized form of gas, which is typicallyassociated with extremely high temperatures. Hot gas plasma, forinstance, is formed on the sun as the result of nuclear fusion. However,cold gas plasma can be formed at low temperatures using low pressureconditions and the right type of energy. Fluorescent lights are anexample. It is this type of gas plasma which is useful for themodification of PHAs. The energy used to create the plasma, such asradio frequency power or electrical discharge, strips electrons from thegas, producing free electrons, gas ions, and excited molecules. When theelectrons recombine with the ions and excited molecules, a glowing“plasma” is produced. Even though the ions and excited molecules mayhave a very high degree of kinetic energy (and thus a high temperature),the temperature of the bulk gas is relatively low (near roomtemperature). These “excited” ions and molecules impact the surface,fragmenting its molecular structure. Recombination events alter thesurface at the molecular level, thereby forming new chemical bonds andintroducing new functional groups. The type of functional group (e.g.,amino, carboxyl, carbonyl, sulfonate, fluorine, or hydroxyl) thatresults depends upon the nature of the plasma gas and the conditions ofthe treatment.

[0046] The PHAs can be treated with a chemical reagent to cleave esterlinkages in the polymer backbone. This results in the formation of freehydroxyl and carboxylic acid groups which alter the charge on thepolymer as well as provides reactive functional groups for subsequentmodification and attachment of molecules. The treatment also can promoteor reduce cellular adhesion by, or cell and tissue growth on, thepolymer. Reagents which can be used to cleave the polymer backboneinclude water, bases, acids, nucleophiles, electrophiles, plasma, andmetal ions. Hydrolysis of the esters can also be performed enzymaticallyusing esterases. The polymers can also be cleaved by irradiation and/orapplication of heat.

[0047] These modifications can be carried out homogeneously in solution.However, if the polymer is in a solid form (e.g., particles or a film),then such modifications may be limited primarily to the polymer surface.This method allows surface properties to be modified without alteringthe overall mechanical properties of the underlying polymer or,accordingly, devices formed of the polymers.

[0048] Certain PHAs with functional pendant groups can also be modifiedby chemical and physical means. Such modifications may change polymerproperties, for example, or allow subsequent attachment of othermolecules or cells. The exact modifications that can be made varyaccording to the nature of the functional group and will be apparent tothose skilled in the art. For example, functional pendant groups likeesters can be converted to acids, and unsaturated groups can be oxidizedto diols, alcohols, aldehydes, and acids. Reagents for modifyingfunctional pendant groups can readily be selected by those skilled inthe art.

[0049] Bioactive species can also be attached to the ends of thepolymers, either covalently or ionically, or by mixing the bioactivespecies with the polymeric material. Coupling chemistry involvinghydroxyl and carboxyl end groups or other reactive groups which can bepresent on the molecules is well known to those skilled in the art.

[0050] The PHAs can also be modified non-covalently. For example, thePHAs include a carboxylic acid group, which can form a ionic bond withamine groups present on materials such as proteins and peptides,polylysine, and other cationic materials. Such modifications can, forexample, change surface properties like hydrophobicity and surfacecharge of the polymers. Examples of molecules which can modify PHAsnon-covalently are surface active agents and lipids.

[0051] 4. Surface Modification of PHAs

[0052] Surface modifications, which introduce new functional groups,allow for the selective attachment of specific bioactive agents. Afterproduction, isolation, and purification of the PHA, the chemical surfacetreatment or gas plasma treatment conditions can be controlled to varythe level of modification. It is also possible to prepare surfacemodification gradients, which allow for the preparation of concentrationgradients of bioactive compounds on a surface. This may be useful forcontrolling tissue regeneration or other processes that are affected bythe concentration of specific agents.

[0053] A significant advantage of a cold plasma treatment is that itaffects all exposed surfaces, as opposed to “line of sight” plasmatreatments or chemical surface treatments. Additionally, plasmatreatment avoids problems, such as “wetting” and residues, associatedwith chemical treatments. Therefore, three dimensional objects may betreated after they are molded or otherwise fabricated, thus minimizingproblems which may arise during processing of functionalized PHAs. Alimitation of gas plasma is that it is not practical for powderymaterials, or liquid suspensions, due to the low pressure conditions ofthe treatment.

[0054] Typical applications for gas plasma involve surface cleaning,surface modification, and polymer deposition. These processes use thesame type of plasma, but differ in the effect on the surface. Cleaningprocesses are designed to remove all organic material from an inorganicmaterial to produce an “atomically clean” surface. Surface modificationof “etching” is used to selectively alter the surface properties of anarticle, while not affecting the bulk of the material. Polymerdeposition is performed to introduce a uniform surface coating to anobject by exposing a plasma activated surface to a polymerizablereagent. Surface modification procedures are expected to be most usefulfor the derivatization of PHAs.

[0055] Oxygen and ammonia gas plasmas can be employed to introduce newcarboxylic or amino groups, respectively, into the polymer. It isbelieved that the oxygen plasma functions as an oxidant. Thesefunctional groups can be utilized as sites for the covalent attachmentof bioactive agents. PHO films have been activated using a gas plasmatreatment, and were subsequently derivatized by covalent attachment ofbioactive agents (see Example 18 which follows).

[0056] In the biomedical field, cold gas plasma treatments are used toincrease biocompatibility, enhance cell attachment, immobilize drugs,reduce allergic response, depyrogenate and sterilize materials ordevices. Treatments can be tailored to meet the specific needs of theapplication.

[0057] 5. Methods for Removing Pyrogens from the PHAs

[0058] The polymers can be purified to reduce pyrogen levels eitherbefore or after fabrication of the polymers into various physical forms,although it is preferable to purify the materials before fabrication. Inthe latex form, the polymer may be subjected to two or more treatmentsdesigned to reduce pyrogen levels. If necessary, the dry solid form canbe reconstituted as a latex using the procedures described, for example,by Koosha, F. Ph.D. Dissertation, 1989, Univ. Nottingham, UK, Diss.Abstr. Int. B 51:1206 (1990). This can be preferred, particularly whenit is desirable to obtain a PHA with very low levels of endotoxin in thepolymer for a tissue engineering application. The PHA latex may be driedto yield a solid form if desired. Preferably, devices prepared from thePHAs have less than 1000 endotoxin units, more preferably, less than 100endotoxin units, and most preferably, less than 20 endotoxin units.

[0059] In the case of a PHA latex, depyrogenation can involve treatmentwith an oxidizing agent. The oxidizing agent must be selected such thatit does not significantly degrade or adversely alter the physical orchemical nature of the PHA. Preferably, the oxidizing agent has goodsolubility in aqueous solutions. A preferred oxidizing agent is hydrogenperoxide. The latex may contain particles of any size, although theparticles are preferably nanoparticles and/or microparticles. The latexparticles may be crystalline or amorphous, but are more preferablyamorphous.

[0060] Solid forms of PHAs, such as powders, films, and pellets, may bedepyrogenated by dissolving the PHA in a suitable organic solvent andthen performing a suitable depyrogenation step. The resulting PHAsolution may be depyrogentaged, for example, with an oxidizing agent.Preferably, the oxidizing agent has good solubility in the organicsolvent used to dissolve the PHA, and does not significantly degrade oradversely alter the physical or chemical nature of the PHA. A preferredoxidizing agent is an organic peroxide. Particularly preferred oxidizingagents for use in organic solvents are aromatic peroxides such asbenzoyl peroxide. Preferred solvents have good solubility for thespecified PHA, and good stability for the oxidizing agent.

[0061] If necessary, the polymers can be depyrogenated by usingcombinations of the aqueous and organic-based oxidation treatments. APHA latex, for example, may first be treated with hydrogen peroxide, andthen subjected to a second treatment in an organic solution with anorganic peroxide to further reduce endotoxin levels.

[0062] While it is generally preferred that a PHA is depyrogenated priorto fabrication of a tissue engineering scaffold or a stent, the methodsdescribed herein may also be applied in whole or in part to depyrogenatea fabricated PHA tissue engineering scaffold or a stent.

[0063] The use of oxidizing agents to reduce endotoxin levels in PHApolymers are preferred to the use of: physical treatments, such as heatand radiation, which can cause polymer degradation; other chemicaltreatments, such as hydrolysis and alkylation, which can modify thepolymer structure; and, filtration and affinity techniques which areeither unsuitable for depyrogenation of latex materials, or mustovercome the high affinity of the PHA for endotoxin. However, these andother depyrogenation methods can be used to obtain the desired purity.

[0064] II. Methods for Manufacturing Medical Devices

[0065] The polymers are useful for preparing a variety of medicaldevices, including biodegradable implants. The biodegradable polymerspreferably exhibit a relatively slow biodegradation, for example, havinga in vivo half-life of between three and six months. The polymerspreferably have a relatively low melting point/glass transitiontemperature, for example, less than 136° C., and/or are soluble in anon-toxic, non-halogenated solvent, for ease of processing.

[0066] When the depyrogenated PHAs are implanted in the body, thesematerials show very little, if any, acute inflammatory reaction or anyadverse tissue reaction. There is no significant inflammatory responseor scar tissue formation. Recruitment of inflammatory cells is minimal.Histological examination of the explanted devices demonstrates that thematerials are essentially inert. Accordingly, devices constructed ofPHAs can be implanted with minimal adverse affect on the surroundingtissue. Release of the hydroxy acid degradation products from theimplanted materials typically is slow and well tolerated by the body.Thus, PHAs are expected to maintain their material properties for amatter of months and will eventually degrade to non-toxic materials.

[0067] Devices prepared from the PHAs can be used for a wide range ofdifferent medical applications. Examples of such applications includecontrolled release, drug delivery, tissue engineering scaffolds, cellencapsulation; targeted delivery, biocompatible coatings; biocompatibleimplants; guided tissue regeneration, wound dressings, orthopedicdevices, prosthetics and bone cements (including adhesives and/orstructural fillers), and diagnostics.

[0068] The PHAs can encapsulate, be mixed with, or be ionically orcovalently coupled to any of a variety of therapeutic, prophylactic ordiagnostic agents. A wide variety of biologically active materials canbe encapsulated or incorporated, either for delivery to a site by thepolyhydroxyalkanoate, or to impart properties to the polymer, such asbioadhesion, cell attachment, enhancement of cell growth, inhibition ofbacterial growth, and prevention of clot formation.

[0069] Examples of suitable therapeutic and prophylactic agents includesynthetic inorganic and organic compounds, proteins and peptides,polysaccharides and other sugars, lipids, and DNA and RNA nucleic acidsequences having therapeutic, prophylactic or diagnostic activities.Nucleic acid sequences include genes, antisense molecules which bind tocomplementary DNA to inhibit transcription, and ribozymes. Compoundswith a wide range of molecular weight can be encapsulated, for example,between 100 and 500,000 grams or more per mole. Examples of suitablematerials include proteins such as antibodies, receptor ligands, andenzymes, peptides such as adhesion peptides, saccharides andpolysaccharides, synthetic organic or inorganic drugs, and nucleicacids. Examples of materials which can be encapsulated include enzymes,blood clotting factors, inhibitors or clot dissolving agents such asstreptokinase and tissue plasminogen activator; antigens forimmunization; hormones and growth factors; polysaccharides such asheparin; oligonucleotides such as antisense oligonucleotides andribozymes and retroviral vectors for use in gene therapy. The polymercan also be used to encapsulate cells and tissues. Representativediagnostic agents are agents detectable by x-ray, fluorescence, magneticresonance imaging, radioactivity, ultrasound, computer tomagraphy (CT)and positron emission tomagraphy (PET). Ultrasound diagnostic agents aretypically a gas such as air, oxygen or perfluorocarbons.

[0070] In the case of controlled release, a wide range of differentbioactive compounds can be incorporated into a controlled releasedevice. These include hydrophobic, hydrophilic, and high molecularweight macromolecules such as proteins. These bioactive compounds mayeither be covalently or non-covalently incorporated. The release profilemay be adjusted by altering one or more of the following parameters: thenature of the PHA; the properties of the bioactive compound; thephysical nature of the drug; and the nature of the device. The phrase“nature of the PHA” is used herein to mean, for example, thecomposition, structure, and molecular weight of the polymer or polymermixture, including crosslinking and crystallinity. The phrase“properties of the compound” is used herein to mean, for example, themolecular weight, hydrophobicity and hydrophilicity. The phrase“physical nature of the compound” is used herein to mean, for example,the particle size and the loading of the compound. The bioactivecompound can be incorporated into the PHAs in a percent loading ofbetween 0.1% and 70% by weight, more preferably between 5% and 50% byweight. The phrase “nature of the device” refers to the device'sphysical shape, thickness, and form, which may be controlled by thefabrication technique.

[0071] PHAs may degrade over a period of as long as five years.Degradability is dependent on properties of the polyhydroxyalkanoate,such as the crystallinity and hydrophobilicty of the polymer, and thesubstitution of the polymer with groups which can promote hydrolysis(such as the copolymerization with polylactic acid, which can decreasedegradation times substantially), as well as the form of the device. ThePHAs may be in almost any physical form, such as a powder, film, moldeditem, particles, spheres, latexes, and crystalline or amorphousmaterials. They can be combined with additional non-PHA materials, forexample, other polymers. They are suitable for use in applicationsrequiring slowly degrading, biocompatible, moldable materials, forexample, medical devices. Examples of medical devices which can beprepared from the polymers include rods, bone screws, pins, surgicalsutures, stents, tissue engineering devices, and drug delivery devices,and wound dressings.

[0072] Degradable implants fabricated with the PHAs may be used in awide range of orthopedic and vascular applications, tissue engineering,guided tissue regeneration, and applications currently served by otherthermoplastic elastomers (McMillin, Rubber Chem. Technol., 67:417-46(1994)). The implants may include other factors to stimulate repair andhealing. Preferred devices are tubes suitable for passage of bodilyfluids. These devices may be modified with cell attachment factors,growth factors, peptides, and antibodies and their fragments.

[0073] 1. General Methods of Preparing Medical Devices

[0074] Preferred methods of fabricating medical devices include solventcasting, melt processing, extrusion, injection and compression molding,and spray drying. Particles are preferably prepared directly from afermentation based process, or by a solvent evaporation technique,double emulsion technique, or by microfluidization, using methodsavailable in the art. (Koosha, F. Ph.D. Dissertation, 1989, Univ.Nottingham, UK., Diss. Abstr. Int. B 51:1206 (1990); Bruhn, B. W. andMüeller, B. W. Proceed. Intern. Symp. Control ReL. Bioact. Mater.18:668-69 (1991); Conti, B. et al., J. Microencapsulation, 9:153-166(1992); Ogawa, Y. et al., Chem. Pharm. Bull., 36:1095-103 (1988);Mathiowitz, E. and Langer, R. “Polyanhydride microspheres as drugdelivery systems,” M. Donbrow Ed., in “Microcapsules Nanopart. Med.Pharm.” CRC, Boca Raton, Fla., 1992, Ch. 5, pp. 99-123.)

[0075] 2. Methods for Fabricating Devices for Wound Healing

[0076] The PHAs can be fabricated into devices suitable for woundhealing. For example, non-woven fibrous materials for this purpose maybe prepared from the polymers by first producing polymer fibers, bypressing the polymers through a perforated outlet, using proceduresknown to those skilled in the art. The fibers can then be fabricatedinto a porous membrane (cloth) by spreading them on a solid support andsubjecting them to compression molding. The thickness of the device ispreferably less than 500 μm. The wound healing device may also beprepared by perforating a film or membrane using a laser to achieveporosity, or using a leaching technique to prepare a porous material.The pore sizes should ideally be small enough to lock out cells andother tissue matter. The wound healing devices may be positioned in vivoto separate tissues and stimulate tissue regeneration.

[0077] 3. Method of Preparing Porous Membranes

[0078] Porous membranes including PHAs may be prepared a variety ofmethods known to those skilled in the art. For example, they may beprepared by solvent casting of polymer solutions containing leachablematerials, and then leaching out the soluble inclusions from thepolymers. Suitable leachable materials are simple non-toxic salts whichdissolve readily in aqueous media. The porosity of the membranes may becontrolled somewhat by selecting leachable materials with differentparticle sizes. After washing, and optionally sterilizing the resultingporous membranes, the membranes may be incubated in cell culture mediaand seeded with cells. Such materials may be used in tissuereconstruction.

[0079] 4. Preparation of Nano or Microparticles

[0080] In one embodiment, nano or microparticles are prepared whichencapsulate one or more agents to be delivered. The particles can beused to locally or systemically deliver a variety of therapeutic agentsto an animal or can be used for diagnostic purposes. Particlesfabricated from PHAs and which encapsulate antigens can be used forimmunization. The preferred particles have particle sizes less than 50μm, more preferably less than 10 μm, and are taken up by the Peyer'spatches when administered orally, and are smaller if for injection.Preferred PHAs for this application are those which can be fabricatedinto vaccine devices without significantly reducing the immunogenicityof the antigen. Preferably, these devices increase the immunogenicity ofthe antigen.

[0081] 5. Cellular Encapsulation

[0082] The PHAs may be used to encapsulate cells. Using procedures knownto those skilled in the art, cells first may be pre-coated. Maysinger,Reviews in the Neurosciences, 6:15-33 (1995). Using a particleencapsulation procedure such as the double emulsion technique, the cellsmay then be encapsulated by PHAs. Ogawa, et al., Chem. Pharm. Bull.,36:1095-103 (1988). Encapsulated cells may then be implanted in vivo.

[0083] 6. Fabrication of PHA Tissue Engineering Scaffolds

[0084] The PHAs may be fabricated into tissue engineering scaffoldsusing a wide range of polymer processing techniques. Preferred methodsof fabricating PHA tissue engineering scaffolds include solvent casting,melt processing, fiber processing/spinning/weaving, extrusion, injectionand compression molding, lamination, and solvent leaching/solventcasting. Such methods are known to those skilled in the art.

[0085] One preferred method of fabricating a PHA tissue engineeringscaffold involves using an extruder, such as a Brabender extruder. Forexample, this technique can be used to prepare extruded tubes suitablefor implantation in a range of lengths and sizes.

[0086] Another preferred method involves preparing a nonwoven PHAscaffold from fibers. Fibers may be produced from the melt or solution,and processed into nonwovens using methods known to those skilled in theart. The properties of the nonwoven may be tailored by varying, forexample, the PHA material, the fiber dimensions, fiber density, materialthickness, fiber orientation, and method of fiber processing.

[0087] Another preferred method of preparing a PHA tissue engineeringscaffold involves using a particulate-leaching technique for preparing ahighly porous membrane. The technique involves dispersing particles in asolution of the PHA polymer, casting the PHA mixture into an appropriatemold, evaporating the solvent, and dissolving the particles out of themembrane. The properties, features and characteristics of the membranemay be varied considerably by altering, for example, the nature and sizeof the particles, applying and using different physical and chemicaltreatments during the fabrication, and varying the PHA type and solventsused. Suitable particles include salt crystals, proteins such as gelatinand agarose, starches, polysaccharides such as alginate and otherpolymers. The diameters of the particles may suitably be betweennanometers to 500 microns. The porous membranes may, if desired, befurther processed. For example, these membranes may be formed intohollow tubes.

[0088] As a variation on the particulate-leaching technique, the PHAscaffolds may be blended with particles, and melt processed into anappropriate mold. Particles may then be leached to yield suitable tissueengineering scaffolds.

[0089] Another preferred method involves melt or solvent processing asuitable PHA into an appropriate mold and perforating the material usinga laser or other means to achieve the desired porosity. Also preferredare methods that include rolling a compression molded PHA sheet into aloop and heat sealing. The PHA sheet optionally may be rolled withanother material, such as a second biodegradable polymer. For example,the latter material could be a nonwoven of polyglycolic acid, polylacticacid, or a copolymer of glycolic and lactic acids. Such a procedureshould provide a laminated tube suitable for use in the engineering ofnew vessels, ducts and tubes.

[0090] The PHAs may also be used to coat other tissue engineeringscaffolds. Such materials could be derived from other degradablepolymers. Coating may be performed, for example, with a solvent basedsolution, or by melt techniques, or using a PHA latex.

[0091] The tissue engineering scaffold may also contain materials otherthan PHAs. These materials may alter, for example, physical and chemicalproperties. Such materials could include plasticizers, nucleatingagents, and other polymers. Moreover, the scaffolds may be fabricated tocontain bioactive compounds, detectable compounds and excipients.Examples of compounds that can be incorporated in PHA tissue engineeringscaffolds include antiplatelet agents such as aspirin, dipyridamole,triclopidine, monoclonal antibody c7E3, integrelin™, MK-852, MK-383,RO-44-9883; antithrombin agents such as heparin, low molecular weightheparin, R-hirudin, hirulog, argatroban, efegatran, Tick anticoagulantpeptide, and Ppack; antiproliferative agents such as angiopeptin,ciprostene, calcium blockers, colchicine, cyclosporine, cytarabine,fusion proteins, Iloprost, ketaserine, prednisone, and trapidil;immunosuppressive agents; factors inhibiting ingrowth of fibrous tissue;factors inhibiting cancerous growth; oligonucleotides such as genes,DNA, and anti-sense sequences; radioactive compounds; growth factors;tissue inducing substances; proteins; peptides; antibodies and antibodyfragments; biopharmaceuticals; and/or other active agents designed topromote, assist, and sustain tissue growth.

[0092] The tissue engineering devices described herein may be seededwith cells prior to implantation or after implantation. The cells may beharvested from a healthy section of the donor's tissue, expanded invitro using cell culture techniques, and then seeded into a scaffold (ormatrix) either prior to or after implantation. Alternatively, the cellsmay be obtained from other donor's tissue or from existing cell lines.

[0093] Examples of cells which can be seeded into tissue engineeringscaffolds include hepatocytes, pancreatic cells, intestinal cells,uroendothelial cells, epithelial cells, skin cells (epidermal cells),muscle cells, nerve cells, mesenchymal cells, myocytes, chondrocytes,adipocytes, fibromyoblasts, ectodermal cells, and bone cells. The cellsmay be genetically engineered. The chosen cells are preferablydissociated, viable, and in suspension prior to application to thescaffold. In the case of scaffolds seeded prior to implantation, thecells should be provided with sufficient time to adhere to the polymerscaffold before being implanted. Alternatively, the PHA tissueengineering device may first be implanted, prevascularized, and thenseeded with cells by, for example, injection.

[0094] The PHAs may be used in tissue engineering applications forvirtually every tissue, including liver, cartilage, kidney, lung, skin,heart, bladder, pancreas, bone, uroepithelial-smooth muscle structures(especially ureters and urethras), tracheal epithelium, tendon, breast,arteries, veins, heart valves, gastrointestinal tubes, fallopian tubes,bile ducts, esophagus, and bronchi.

[0095] It may also be desirable to use the tissue engineering materialsin conjunction with other therapies such as gene therapy, radiotherapy,and therapies requiring localization or delivery of an active agent.Tissue engineering may, for example, be used to deliver factorsexpressed by cells, which can if desired be genetically engineered, forthe treatment of diseases.

[0096] 7. Use of PHAs to Coat Devices

[0097] The PHAs may be used to coat other devices and materials. Suchcoatings may improve their properties for medical application, forexample, improving their biocompability, mechanical properties, andtailoring their degradation and controlled release profiles. The PHAsmay be coated onto other devices using the fabrication proceduresdescribed above. The thickness of the coating can be adjusted to theneeds of the specific application by changing the coating weight orconcentration applied, and/or by overcoating.

[0098] 8. Fabrication of PHA Stents

[0099] The PHAs may be fabricated into stents using a wide range ofpolymer processing techniques. Preferred methods of fabricating PHAstents include solvent casting, melt processing, fiberprocessing/spinning, extrusion, injection molding, and compressionmolding. Such methods are known to those skilled in the art.

[0100] One preferred method of fabricating a stent involves extrudingsmall tubes using, for example, a Brabender™ extruder. The tubes may bemade in a range of lengths and sizes. Preferably the tubes should havesmooth surfaces to provide good compatibility and fit against vesselwalls. If desired the tubes may be perforated, for example, using alaser or by other means.

[0101] Another preferred method involves rolling a compression moldedPHA sheet into a loop and heat sealing. The PHA sheet may optionally berolled with another material, such as a second biodegradable polymer.The latter could, for example, be a polylactic acid mesh. Such aprocedure provides a laminated stent.

[0102] The PHAs may also be used to coat existing stent materials. Suchmaterials could be metallic, non-degradable polymers, or other non-PHAdegradable polymers. Coating may be performed, for example, with asolvent based solution, or by melt techniques, or using a PHA latex.

[0103] The stent may also contain materials other than PHAs. Thesematerials may alter, for example, physical and chemical properties. Suchmaterials could include plasticizers, nucleating agents, and otherpolymers. Moreover, the devices may be fabricated to contain bioactivecompounds, detectable compounds and excipients. Examples of compoundswhich may be included in PHA stents are antiplatelet agents such asaspirin, dipyridamole, triclopidine, monoclonal antibody c7E3,integrelin, MK-852, MK-383, RO-44-9883; antithrombin agents such asheparin, low molecular weight heparin, R-hirudin, hirulog, argatroban,efegatran, Tick anticoagulant peptide, and Ppack; antipoliferativeagents such as angiopeptin, ciprostene, calcium blockers, colchicine,cyclosporine, cytarabine, fusion proteins, Iloprost, ketaserine,prednisone, and trapidil; radioactive compounds; and oligonucleotidessuch as genes and anti-sense sequences. In addition, the PHA stents alsomay contain cells, particularly genetically engineered cells, viruses,and other therapeutically beneficial components.

[0104] 9. Targeting of PHA Particles and Devices

[0105] The PHAs may be modified for passive targeting after fabricationas devices or particles. Optionally, the particles may contain bioactivecompounds or detectable substances. Preferred particle sizes are lessthan 100 μm, more preferably less than 15 μm. The particles may befurther modified to improve targeting and prevent removal from thecirculation by covalent or non-covalent incorporation of additionalmolecules. Examples of molecules which may be incorporated to improvetargeting and prevent removal from the circulation are surface activeagents, charged molecules, and PEG.

[0106] In addition to passive targeting, the PHAs may be activelytargeted to a particular organ, group of cells within an organ, or sitewithin a cell. Alternatively, an implant device prepared from lowmelting PHAs may actively attract bioactive substances, including cells.Using the procedures described for covalent modification of low meltingPHAs and their derivatives, and those procedures described forfabrication of PHA devices, targeting molecules may be attached to thepolymers. For example, targeting sequences such as peptides andproteins, such as antibodies and their fragments, may be coupled tocarboxylic acids liberated by partial hydrolysis of the polymer backboneor present on the polymer's pendant groups. Other coupling chemistry,known to those skilled in the art, can be used to covalently attachthese targeting sequences and others to different functional groups onthe PHAs.

[0107] Preferred functional groups for attaching of targeting moleculesto PHAs are carboxylic acids, amines, thiols, alcohols, unsaturatedgroups, and halogens. Preferred targeting molecules are cell attachmentfactors, antibodies, antibody fragments, and growth factors. The devicesprepared from low melting PHAs containing active targeting molecules maybe injected, implanted or delivered orally, and used, for example, inwound healing applications such as guided tissue regeneration, andchemotherapy.

[0108] 10. Sterilization

[0109] Prior to implantation, a bioresorbable polymeric article must besterilized to prevent disease and infection of the recipient.Sterilization is performed prior to seeding a polymeric device withcells. Heat sterilization of PHA containing articles is oftenimpractical since the heat treatment could deform the article,especially if the PHA has a melting temperature below that required forthe heat sterilization treatment. This problem can be overcome usingcold ethylene oxide gas as a sterilizing agent. Exposure of a PHAcontaining article to vapors of ethylene oxide prior to implantationsterilizes the article making it suitable for implantation. Duringsterilization with coldethylene oxide gas, the PHA containing articlemaintains its shape. This type of treatment is ideally suited forsterilization of molded, or pre-formed articles where the shape of thearticle plays in important role in its proper functioning.

[0110] Ethylene oxide vapors should also aid in the detoxification ofpyrogen contaminants of a PHA containing article. As a powerfulalkylating agent, ethylene oxide can detoxify pyrogens through analkylation mechanism. This mechanism of detoxification is similar tothat of acylation by an anhydride or mixed anhydride. For example,treatment of pyrogens or endotoxins with acetic anhydride or succinicanhydride is a well known method for detoxification and depyrogenation.The mechanism of action for detoxification of pyrogens by theseanhydrides is believed to be conversion of the pyrogens to a non-toxicderivative via acylation. Ethylene oxide will behave in a similar mannervia alkylation of the pyrogens.

[0111] III. Methods of Using Articles of Manufacture

[0112] The devices described herein can be administered systemically orlocally, or even used in vitro, particularly for cell culture. Thepreferred methods of systemically administering the devices are byinjection, inhalation, oral administration and implantation. Othersuitable methods for administering the devices include administering thedevices topically, as a lotion, ointment, patch, or dressing. Thepolymers can also be incorporated into chewing gums, a technique knownto those skilled in the art. Rassing, Adv. Drug Delivery Rev., 13:89-121(1994). The PHAs devices prepared according to the above procedures canbe used for a wide range of different medical applications.

[0113] The compositions and methods described herein will be furtherunderstood with reference to the following non-limiting examples.

EXAMPLE 1 Depyrogenation of a Copolymer Latex of R-3-hydroxyoctanoicAcid and R-3-hydroxyhexanoic Acid With Hydrogen Peroxide

[0114] A copolymer of R-3-hydroxyoctanoic acid and R-3-hydroxyhexanoicacid, derived by fermentation, containing over one million endotoxinunits per gram (EU/gram), as measured with the copolymer in a latexform, was depyrogenated.

[0115] The latex was prepared by dissolving hexane-extracted PHA inacetone (1% wt/vol, 2.5 mL) and adding via flame-drawn pipet into 5 mLpyrogen-free water at 80° C. After 30 min at this temperature, thesample was divided into two equal portions: control and treated. To thetreated sample was added 25 microliters 50% hydrogen peroxide and thesample was boiled for one hour. Both control and treated samples wereassayed for endotoxin by the Limulus amebocyte lysate (LAL) test(Associates of Cape Cod, Mass.).

[0116] The endotoxin content of the polymer after treatment of thepolymer was less than 6 EU/gram.

EXAMPLE 2 Depyrogenation of a Solid Copolymer of R-3-hydroxyoctanoicAcid and R-3-hydroxyhexanoic Acid With Hydrogen Peroxide

[0117] A copolymer of R-3-hydroxyoctanoic acid and R-3-hydroxyhexanoicacid, derived by fermentation, containing over one million EU/gram, asmeasured by the LAL test, was depyrogenated by treating the bulk polymerwith aqueous hydrogen peroxide at 80° C. in a biphasic reaction.

[0118] The endotoxin content of the polymer after treatment of thepolymer was 100 EU/gram, measured as a latex using the LAL test.

EXAMPLE 3 Depyrogenation of a Solid Copolymer of R-3-hydroxyoctanoicAcid and R-3-hydroxyhexanoic Acid With Phthalic Anhydride

[0119] A copolymer of R-3-hydroxyoctanoic acid and R-3-hydroxyhexanoicacid, derived by fermentation, containing over one million EU/grammeasured by the LAL test, was depyrogenated by treating the bulk polymerwith phthalic anhydride at 75° C.

[0120] The endotoxin content of the polymer after treatment was 500EU/gram, measured as a latex using the LAL test.

EXAMPLE 4 Depyrogenation of a Solution of the Copolymer ofR-3-hydroxyoctanoic acid and R-3-hydroxyhexanoic Acid With PotassiumPermanganate

[0121] A copolymer of R-3-hydroxyoctanoic acid and R-3-hydroxyhexanoicacid, derived by fermentation, containing over one million EU/grammeasured by the LAL test, was depyrogenated by treating the polymerdissolved in dichloromethane with potassium permanganate.

[0122] The endotoxin content of the polymer after treatment of thepolymer was 2500 EU/gram, measured as a latex using the LAL test.

EXAMPLE 5 Depyrogenation of a Solution of the Copolymer ofR-3-hydroxyoctanoic Acid and R-3-hydroxyhexanoic Acid With SodiumHypochlorite

[0123] A copolymer of R-3-hydroxyoctanoic acid and R-3-hydroxyhexanoicacid, derived by fermentation, containing over one million EU/grammeasured by the LAL test, was depyrogenated by treating the polymerdissolved in dichloromethane with sodium hypochlorite.

[0124] The endotoxin content of the polymer after treatment of thepolymer was 8000 EU/gram, measured as a latex using the LAL test.

EXAMPLE 6 Depyrogenation of a Solution of the Copolymer ofR-3-hydroxyoctanoic Acid and R-3-hydroxyhexanoic Acid With Basic Alumina

[0125] A copolymer of R-3-hydroxyoctanoic acid and R-3-hydroxyhexanoicacid, derived by fermentation, containing over one million EU/grammeasured by the LAL test, was depyrogenated by treating the polymerdissolved in dichloromethane with basic alumina.

[0126] The endotoxin content of the polymer after treatment of thepolymer was 8000 EU/gram, measured as a latex using the LAL test.

EXAMPLE 7 Depyrogenation of a Solution of the Copolymer ofR-3-hydroxyoctanoic Acid and R-3-hydroxyhexanoic Acid With BenzoylPeroxide

[0127] A copolymer of R-3-hydroxyoctanoic acid and R-3-hydroxyhexanoicacid, derived by fermentation, containing over one million EU/grammeasured by the LAL test, was depyrogenated by treating the polymerdissolved in dichloromethane with benzoyl peroxide. The PHA (0.25 g) wasdissolved in dichloromethan (2.5 mL), to which was added benzoylperoxide (50 mg) and stirred overnight at room temperature. The reactionmixture was then filtered through a cotton-plugges Pasteur pipettecontaining 2 g basic alumina to remove residual peroxide. The polymerwas recovered from the filtrate by rotary evaporation and then preparedas an artificial latex and assayed for endotoxin.

[0128] The endotoxin content of the polymer after treatment of thepolymer was 4000 EU/gram, measured as a latex using the LAL test.

EXAMPLE 8 Depyrogenation of Whole Cells Including a Copolymer ofR-3-hydroxyoctanoic Acid and R-3-hydroxyhexanoic Acid With HydrogenPeroxide

[0129] A suspension of whole cells including a copolymer ofR-3-hydroxyoctanoic acid and R-3-hydroxyhexanoic acid, derived byfermentation, containing over one million EU/gram measured by the LALtest, was depyrogenated by treatment with aqueous hydrogen peroxide (2%peroxide, 0.6% surfactant, 10% solids, pH 7, 80° C.) for 3.5 hours.

[0130] After treatment, the composition was cooled, centrifuged, washedwith an aqueous surfactant solution, water, precipitated, pelleted bycentrifugation, and freeze dried. The endotoxin content of the treatedpolymer was 50 EU/gram, measured as a latex using the LAL test.

EXAMPLE 9 Depyrogenation of Whole Cells IncludingPoly-R-3-hydroxybutyric Acid With Hydrogen Peroxide

[0131] A suspension of whole cells including poly-R-3-hydroxybutyricacid (PHB), derived by fermentation, was depyrogenated by treatment withaqueous hydrogen peroxide (2% peroxide, 0.6% surfactant, EDTA, 10%solids, pH 7, 80° C.) for 3.5 hours. After treatment, the compositionwas cooled, centrifuged, washed with an aqueous surfactant solution,water, precipitated, pellet by centrifugation, and freeze dried.

[0132] The endotoxin content of the treated polymer was less than 0.12EU/gram, measured as a powder using the LAL test. By comparison theendotoxin content of a commercial sample of PHB was greater than 120EU/gram measured as a powder.

EXAMPLE 10 Method for Preparing a High Purity Polyhydroxyalkanoate Witha Melting Point Below 136° C. Using Chloroform Extraction

[0133] A copolymer of R-3-hydroxyoctanoic acid and R-3-hydroxyhexanoicacid with a melting point of 61° C., derived by fermentation ofPseudomonas putida KT2442, was extracted from freeze-dried cells withchloroform. The chloroform solution was filtered through a glassmicrofiber filter (2.7 μm) to remove particulates. The solution wasconcentrated, and the polymer precipitated via slow addition of a tenfold excess of methanol. The polymer was then dissolved in chloroformand cast as a film. The chloroform was allowed to evaporate completely,to yield a polymeric film.

EXAMPLE 11 Method For Preparing a High Purity Polyhydroxyalkanoate Witha Melting Point Below 136° C. Using Hexane Extraction

[0134] A copolymer of R-3-hydroxyoctanoic acid and R-3-hydroxyhexanoicacid with a melting point of 61° C., derived by fermentation ofPseudomonas putida KT2442, was extracted from freeze-dried cells withhexane. The hexane solution was filtered through a glass microfiberfilter (2.7 μm) to remove particulates. The hexane was removed bydistillation. The polymer was dissolved in acetone, and the polymerprecipitated by addition of a ten fold excess of methanol. The polymerwas collected, dissolved in acetone, and cast as a film. The acetone wasallowed to evaporate completely to yield a polymeric film.

EXAMPLE 12 Method For Preparing a High Purity Polyhydroxyalkanoate Witha Melting Point Below 136° C. Using Acetone Extraction

[0135] A copolymer of R-3-hydroxyoctanoic acid and R-3-hydroxyhexanoicacid with a melting point of 61° C., derived by fermentation ofPseudomonas putida KT2442, was extracted from freeze-dried cells withacetone. The acetone solution was filtered through a glass microfiberfilter (2.7 μm) to remove particulates. The acetone was removed bydistillation. The polymer was dissolved in acetone, and the polymerprecipitated by addition of water. The polymer was collected usingcentrifugation to assist in the separation of phases, dissolved inacetone, and cast as a film. The acetone was allowed to evaporatecompletely to yield a polymeric film.

EXAMPLE 13 Method For Preparing a High Purity Polyhydroxyalkanoate LatexParticles With a Melting Point Below 136° C.

[0136] Whole cells containing a copolymer of R-3-hydroxyoctanoic acidand R-3-hydroxyhexanoic acid with a melting point of 61° C., derived byfermentation of Pseudomonas putida KT2442, were treated with 2% aqueoushydrogen peroxide, SDS (0.6%), pH 7, 80° C., for 3.5 hours. The latexwas cooled, centrifuged and washed three times with a 0.4% SDS solution,and then washed two times with water.

EXAMPLE 14 Biocompatibility of a Polyhydroxyalkanoate PolymerComposition With a Melting Point Below 136° C.

[0137] A copolymer of R-3-hydroxyoctanoic acid and R-3-hydroxyhexanoicacid with a melting point of 61° C., derived by fermentation, wasfabricated into devices and implanted subcutaneously in adult femalemice. The implants were removed at 2, 4, 8, 12 and 40 weekspost-implantation and prepared for histological examination.Biocompatibility of the implants was evaluated by determining the degreeof the fibrotic reaction surrounding the tissue samples and the presenceof inflammatory cells.

[0138] Histological analysis showed minimal tissue reaction with nomacrophages or histocytes present, suggesting that there were no signsof chronic inflammation or foreign body response to thepolyhydroxyalkanoate.

EXAMPLE 15 In Vivo Degradation of a Polyhydroxyalkanoate PolymerComposition With a Melting Point Below 136° C.

[0139] The molecular weight of the copolymer of Example 10 wasdetermined prior to implantation, and after being implanted for 40weeks. A control unimplanted sample was checked after 40 weeks.Molecular weight was determined by GPC.

[0140] The weight average molar mass, Mw, of the polymer prior toimplantation was 137,000. After 40 weeks, although there were no visiblesigns of degradation or loss of mechanical properties, the Mw of thepolymer was around 65,000. The number average molar mass, Mn, of thepolymer prior to implantation was 58,000 compared to 31,000 after 40weeks of implantation. Additional samples were taken from implants tocompare the molecular weights at the surface versus the interior of theimplants. No significant differences were observed, indicating a slowhomogeneous hydrolytic breakdown of the polymer in vivo.

EXAMPLE 16 Controlled Release From a Device Prepared From Low MeltingPHAs

[0141] Controlled release devices were fabricated from a copolymer ofR-3-hydroxyoctanoic acid and R-3-hydroxyhexanoic acid with a meltingpoint of 61° C. Model drug and polymer were co-dissolved in chloroformat 20% wt/vol total concentration and cast in small Petri dishes. Afterstanding in air for several days, polymer films were obtained whichcontained the drug in dispersed form. Disks were then cut from each filmsample using a {fraction (7/16)}″ cork borer. The devices containedthree different compounds, which (in order of increasing lipophilicity)were 3-hydroxytheophylline, prednisolone-21-hemisuccinate, andlidocaine. Each drug was tested at three different loadings (4, 10, and25% wt/wt). Each device, typically weighing around 85 mg, was depositedin a stoppered glass tube and immersed in 2 mL buffer comprising 100 mMsodium phosphate, pH 7.4, 0.02% sodium azide (sterile filtered) andmaintained at 37° C. A control device containing polymer alone wasincluded in the studies to monitor degradation of the polymer andrelease of impurities. At regular intervals, a sample was withdrawn andreplaced with an equal volume of fresh buffer. Release of the compoundwas monitored by UV spectroscopy at the appropriate wavelengths(β-hydroxytheophylline [l=272 nm], prednisolone-21-hemisuccinate [l=252nm], and lidocaine [l=262 nm]), and release values were normalizedagainst the total quantity of drug released over the study period. Forall the devices a significant amount of the compound was released in thefirst 24 hour period. After this, for both β-hydroxytheophylline andlidocaine, the percent of drug release on a daily basis was inverselyproportional to the percentage of drug loading.

EXAMPLE 17 Chemical Modification of PHO Film

[0142] A PHO film was dotted with DMSO solutions of5-(Biotinamido)-pentylamine (Pierce Chemical Col. product #212345) in avariety of organic co-solvents: isopropanol, acetonitrile, methanol,ethanol, and propylene carbonate. The biotin reagent contains a primaryamino group attached to a pentamethylene linker. This amino group cancause aminolysis reactions with the polyester backbone of the PHA,resulting in scission of the polymer chain and covalent attachment ofbiotin. After biotinylation, the excess reagent was washed away and theamount of biotinylation was quantified using ELISA (enzyme linkedimmunosorbent assay) techniques with a streptavadinhorseradishperoxidase/chemiluminescence system. The strength of the signal, whichis proportional to the amount of biotin, was dependent upon thecosolvent used and increased in the series: isopropanol greater thanacetonitrile which is greater methanol, ethanol greater than propylenecarbonate. Solvent controls were performed to demonstrate the dependenceof the signal on the biotin reagent.

EXAMPLE 18 Gas Plasma Modification of PHO Film

[0143] PHO films were gas plasma treated in two different plasmas,ammonia and oxygen. These plasmas are expected to introduce new aminoand carboxyl functional groups, respectively. These functional groupscan act as sites for the covalent attachment of bio-active agents, suchas a protein. Surface treatment was found to increase the waterwettability of the PHO film compared with untreated PHO. This result isa strong indication of surface modification. Protein (rabbit IgG) wascovalently coupled to the films using an aqueous solution of the proteinand the coupling reagent EDC. The amount of coupled protein wasquantified using a goat anti-rabbit antibody/horseradishperoxidase/chemiluminescence system. The strength of the signal wasfound to be proportional to the amount of rabbit IgG attached to thesurface. Controls (-EDC, -rabbit IgG) were performed to demonstrate thedependence of the signal on these two components.

EXAMPLE 19 Tubular Tissue Engineering Devices

[0144] Tissue engineered tubular devices can serve a variety offunctions for the replacement of tubular, biological tissues, such asvascular grafts, heart valves, urethra, intestines, nerve growth tubes,ducts, sphincter muscles, skin sheathes, etc. As PHAs aresimi-crystalline, thermoplastic materials, a tubular PHA device can beformed in a variety of ways, such as extrusion, molding, pressing, andshaping or from solution using solution casting techniques. Afterforming the PHA into the desired shape and allowing the PHA tocrystallize, a PHA article will maintain its shape for use as a tissueengineered article.

[0145] PHO was pressed into a thin film between two sheets of Mylar™. ACarver press at a platen temperature of 60° C. was used to supply 1 tonof pressure for 20 seconds. Spacers of appropriate thickness are used tocontrol film thickness. The pressed film was placed in a refrigerator at4° C. overnight to allow the PHO to crystallize. The PHO film wasremoved from the Mylar™ backing sheets and rolled into a tube shape on acylindrical, Teflon™ support. The support diameter was chosen to producea tube of a desired size and diameter. The film edges at the seam weresealed using compression, however, the seams may also be sealed viawelding, melting, or partially dissolving the edges together. The tubewas allowed to solidify prior to removal from the support.

EXAMPLE 20 Porous Tissue Engineering Devices

[0146] It is desirable to utilize a porous material for many tissueengineering applications. There are several advantages to using a porousmaterial such as better diffusion of fluids and nutrients, increasedsurface area, increased cellular attachment, faster degradation, andgreater tissue contact. For many tissue engineering applications, it isdesired to utilize pores which are approximately 50 to 200 μm indiameter (for seeding of cells, preferred interstitial spacings on theorder of 100 to 300 microns are not unusual), however, the optimumporosity, pore size and density of a porous material will vary dependingupon its intended application. Pores can be introduced in a polymericmaterial using a variety of techniques such as foaming agents,processing of fibers into woven or non-woven structures, phaseseparation and leaching. Leaching strategies involve dispersing a solidmaterial (such as salt) within the polymer. The solid material is chosensuch that it is poorly soluble in the polymer and readily removed byleaching. The solid can be an inorganic or organic material, forexample, a salt, sugar, protein, or polymer. After dispersing the solidin the polymer, the mixture can be formed into the desired shape. Afterconstructing a device from the solid-polymer mixture, the solid isselectively dissolved away using a solvent in which the solid is solublebut in which the polymer is poorly soluble. The solid particles dissolveaway to leave behind vacant pores. The size, distribution, and weightpercent of the particles may be chosen to produce materials with a rangeof porosities.

[0147] A porous PHA tube was made. PHO was melted and mixed with sievedsalt particles in a weight ratio of 1 to 2 to yield a homogeneousmixture. The salt particles used had been sieved between 80 and 180 μm,however the particle size, distribution and weight percent may be varieddepending upon the desired pore size and density. The PHO/salt mixturewas pressed into a thin film between two sheets of Mylar™. A Carverpress at a platen temperature of 60° C. was used to supply 1 ton ofpressure for 20 seconds. Spacers of appropriate thickness were used tocontrol film thickness. The pressed film was placed in a refrigerator at4° C. overnight to allow the PHO to crystallize. The PHO/salt film wasremoved from the Mylar™ backing sheets and rolled into a tube shape on acylindrical, Teflon™ support. The support diameter was chosen to producea tube of a desired size and diameter. The film edges at the seam weresealed using compression, however, the seams may also be sealed viawelding, melting, or partially dissolving the edges together. The tubewas allowed to solidify and was soaked in a water bath with frequentchanges of the water to dissolve away the salt. After exhaustiveleaching of the salt, a porous tube of PHO remained. The porous tube hadproperties which make it suitable for use as a vascular graft.

EXAMPLE 21 Construction of a PHA Heart Valve

[0148] Tissue engineered PHA devices can serve a variety of functionsfor the replacement of complex biological tissues, including valves,organs, and skeletal tissues. As PHAs are semi-crystalline,thermoplastic materials, a PHA device can be formed in a variety of wayssuch as extrusion, molding, pressing, and shaping or from solution usingsolution casting techniques. After forming the PHA into the desiredshape and allowing the PHA to crystallize, a PHA article will maintainits shape for use as a device for tissue engineering.

[0149] A porous PHA heart valve was made. PHO was melted and mixed withsalt particles in a weight ratio of 1 to 2 to yield a homogeneousmixture. The salt particles had been sieved between 80 and 180 μm,however, the particle size, distribution and weight percent may bevaried depending upon the desired pore size and density. The PHO/saltmixture was pressed into a thin film between two sheet of Mylar™. ACarver press at a platen temperature of 60° C. was used to supply 1 tonof pressure for 20 seconds. Spacers of appropriate thickness were usedto control film thickness. The pressed film was placed in a refrigeratorat 4° C. overnight to allow the PHO to crystallize. The PHO/salt filmwas removed from the Mylar™ backing sheets. A PHO/salt film ofapproximately 250 μm thickness was used to form each of three valveleaflets. The leaflets were cut into the desired shape, whichapproximated that of biological leaflet. The leaflets were welded ontoanother PHO/salt film which served as a cylindrical conduit. The conduitfilm was 1 mm thick and made from a PHO/salt mixture of the samecomposition as the leaflets. Welding was performed using a heated probeat 50° C. to melt the PHO together at the seams. The leaflets werepositioned using a natural heart valve as a model. The conduit waswelded into the shape of a tube to complete the construction of thevalve. The valve was allowed to solidify at 4° C. overnight. Afterexhaustive leaching of the salt in a water bath, a porous PHO heartvalve remained. The valve leaflets had very good flexibility and thevalve had very good handleability.

EXAMPLE 22 Cell Seeding of PHA Materials

[0150] Prior to implantation, tissue engineered materials may be seededwith cells to enhance their biocompatibility and/or to promote thegrowth of a desired tissue. The cells used are chosen from theappropriate type of tissue and are preferentially harvested from thepatient to minimize tissue rejection, however, the cells may come from acell bank. Additionally, bioactive compounds which direct the growth ofa desired tissue, such as cell attachment proteins, can be incorporatedonto or into a tissue engineered material prior to cell seeding andimplantation. The tissue engineering material thus serves as a solidsupport to organize the cells for proper growth. After cell seeding, thecells can be grown in vitro on the tissues construct to reach thedesired cell density.

[0151] A porous PHO sample was sterilized in ethylene oxide gas. Thepolymer was seeded with bovine endothelial cells in fetal bovine serumand then incubated in vitro at 37° C. with 5% carbon dioxide. After 24hours, the cells were fixed onto the sample. Microscopic examination ofthe sample demonstrated good cellular attachment and biocompatibility.

EXAMPLE 23 Surface Modification of a PHA Film

[0152] The surface properties of a tissue engineering device are veryimportant, since the surface is the interface between the host's livingtissue and the implanted device. Surface modification can introduce newfunctionality to a polymer surface without significantly modifying thebulk properties of the polymer. Some surface properties which can bemodified include hydrophobicity, hydrophilicity, wettability, cellularattachment, and surface charge. It is preferred to adjust the surfaceproperties of a device to suit its intended application. For instance,often it is preferred to maximize cellular attachment to a device. Insuch a case, the surface of the device might be coated with a bioactivecompound or peptide which promotes cellular attachment, such asfibronectin, laminin or gelatin. These bioactive compounds may becovalently or non-covalently attached to the surface, depending upon theapplication.

[0153] Gas plasma treatment was used to modify the surface of a PHAfilm. In order to prevent sample melting during treatment, conditionswere designed to keep heating to a minimum. A PHO film was treated withan ammonia gas plasma, at 250 microns of ammonia with a flow of 350 SCCMwith 220 watts of power for 10 minutes. Typically, plasma treatmentcovalently modifies a material's surface. After treatment, surfacemodification was confirmed by ESCA analysis. Stable incorporation ofabout 8% nitrogen was achieved. After treatment, samples were stored at4° C. and RT. Surface wettability was determined by water contact anglemeasurements. Untreated PHO has a high contact angle (approximately95°). After treatment the contact angle decreased dramatically to about20 to 30°, demonstrating an increase in wettability. The contact anglesof the treated and untreated samples were stable over at least 30 days,demonstrating stability of the surface modification. ESCA analysis wasrepeated after thirty days and confirmed stability of the surfacemodification.

EXAMPLE 24 Attachment of Bioactive Materials to PHA

[0154] A device used for tissue engineering may have incorporated insideof it or onto its surface a bioactive compound(s). Representativecompounds include growth factors to stimulate tissue growth, cellularattachment proteins to promote tissue attachment, or anti-coagulants toprevent thrombogenesis. Additionally, compounds may be included whichreduce the immune response, label the material for later retrieval,enhance biocompatibility, deliver drugs or the like.

[0155] A bioactive compound was attached to a PHA surface, as follows. APHO film was modified with a biologically active compound, biotin, afterammonia gas plasma treatment. An aqueous solution containing anactivated form of biotin was applied to the treated PHO film. The biotinderivative contained an N-hydroxysuccinimide ester functional groupwhich activated it for acylation. After treatment with biotin-NHS, thefilm surface was washed with water, quenched with glycine and blockedwith 0.1% gelatin. Detection with a strepavidin horseradish peroxidaseconjugate and a chemiluminescent HRP detection solution demonstratedbiotin modification of the surface. A PHO film without ammonia gasplasma treatment was used as a control and demonstrated no biotinmodification under identical conditions.

EXAMPLE 25 Testing of a Hypoallergenic Latex

[0156] A 60 cm² sample of PHO was assayed for skin sensitizationaccording to standard test method ASTM F720. The sample, 3 mm inthickness, was extracted with saline at 50° C. for 72 hr (20 ml). Guineapigs, previously subjected to two-stage induction (21 days) were exposedon the skin to patches soaked in the saline extract, pure saline, or apositive control solution (5% oxazolone in acetone). After 24 hrs, thepatches were removed, and the guinea pigs were checked for a skinreaction after 1, 24 and 48 hours. There was no discernibleerythema/eschar formation with the PHO extract or the pure saline;positive controls all showed well-defined to severe erythema/escharformation within one hour.

[0157] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

We claim:
 1. A polyhydroxyalkanoate polymer composition which does notelicit an acute inflammatory response when implanted into an animal. 2.The composition of claim 1 wherein the polyhydroxyalkanoate compositionis prepared using a process which incorporates a pyrogen into thepolyhydroxyalkanoate, and pyrogen has been removed until thepolyhydroxyalkanoate does not elicit an acute inflammatory response whenimplanted into an animal.
 3. The composition of claim 1 wherein thepolyhydroxyalkanoate is prepared by bacterial fermentation.
 4. Thecomposition of claim 3 , wherein the bacteria is a Gram negativebacteria and the pyrogen is endotoxin, and the polyhydroxyalkanoatecontains less than 100 units of endotoxin.
 5. The composition of claim 4wherein the polyhydroxyalkanoate contains less than 20 units ofendotoxin.
 6. The composition of claim 1 wherein pyrogen is removed byexposing the polyhydroxyalkanoate to an effective amount of an oxidizingagent.
 7. The composition of claim 6 wherein the oxidizing agent is aperoxide.
 8. The composition of claim 1 wherein the polyhydroxyalkanoateincludes at least one monomeric unit of the formula—OCR¹R²(CR³R⁴)_(n)CO— wherein n is an integer between one and 15; andwherein R¹, R², R³, and R⁴ are independently selected from the groupconsisting of hydrogen, methyl, C₂₋₁₅ straight, branched or cyclicalkyl, alkenyl or alkynyl groups, alkaryl groups, aralkyl groups,heteroalkyl groups, heteroaryl groups, hydroxy groups, thiol groups,disulfides, ether groups, thiolether groups, ester groups, carboxylicacid groups, amine groups, amide groups, halogens, nitrogen-substitutedradicals; and oxygen-substituted radicals.
 9. The composition of claim 8wherein the polyhydroxyalkanoate is chemically modified or derivatized.10. The composition of claim 9 wherein an attachment or targetingmolecule is covalently coupled to the polyhydroxyalkanoate.
 11. Thecomposition of claim 1 wherein the polymer is a polyhydroxyalkanoateblend or copolymer.
 12. The composition of claim 1 wherein thepolyhydroxyalkanoate has a melting point or glass transition temperatureless than 136° C.
 13. The composition of claim 1 as an aqueous latexformulation.
 14. A method for producing a biocompatiblepolyhydroxyalkanoate polymer composition comprising: selecting apolyhydroxyalkanoate composition which is prepared using a process whichincorporates a pyrogen into the polyhydroxyalkanoate, and removingpyrogen from the polyhydroxyalkanoate until the polyhydroxyalkanoatedoes not elicit an acute inflammatory response when implanted into ananimal.
 15. The method of claim 14 wherein pyrogen is removed byexposing the polyhydroxyalkanoate to an effective amount of an oxidizingagent.
 16. The method of claim 14 wherein the oxidizing agent is aperoxide.
 17. A method of forming a biocompatible medical devicecomprising selecting a polyhydroxyalkanoate which is prepared using aprocess which incorporates a pyrogen into the polyhydroxyalkanoate, fromwhich the pyrogen has been removed so that the polyhydroxyalkanoate doesnot elicit an acute inflammatory response when implanted into an animal,and forming the device.
 18. The method of claim 17 wherein the pyrogenlevel is less than 100 endotoxin units (EU) per device.
 19. The methodof claim 17 wherein the pyrogen level is less than 20 endotoxin units(EU) per device.
 20. The method of claim 17 wherein the polymer includesat least one monomeric unit of the formula —OCR¹R²(CR³R⁴)_(n)CO— whereinn is an integer between one and 15; and wherein R¹, R², R³, and R⁴ areindependently selected from the group consisting of hydrogen, methyl,C₂₋₁₅ straight, branched or cyclic alkyl, alkenyl or alkynyl groups,alkaryl groups, aralkyl groups, heteroalkyl groups, heteroaryl groups,hydroxy groups, thiol groups, disulfides, ether groups, thiolethergroups, ester groups, carboxylic acid groups, amine groups, amidegroups, halogens, nitrogen-substituted radicals, and oxygen-substitutedradicals, and the polymer has been chemically modified.
 21. The methodof claim 17 wherein the device is in a form selected from the groupconsisting of stents, coatings on prosthetic devices, sutures, staples,and tubing.
 22. The method of claim 17 wherein the device is in a formselected from the group consisting of tissue regeneration devices, cellculture devices, wound dressings, and cell or tissue coatings.
 23. Themethod of claim 17 wherein the device is a porous membrane.
 24. Themethod of claim 17 wherein the device is in the form of microparticlesor nanoparticles.
 25. The method of claim 17 wherein the devicecomprises a material selected from the group consisting of therapeutic,prophylactic and diagnostic agents.
 26. The method of claim 25 , whereinthe therapeutic agent is selected from the group consisting of peptidesand proteins, nucleic acids, saccharides and polysaccharides, lipids,synthetic drug molecules, and imaging agents.
 27. The method of claim 25wherein the device is formulated for administration to a mucosalsurface.
 28. The method of claim 17 wherein the polyhydroxyalkanoate isa polymer blend or copolymer.
 29. The method of claim 28 wherein thepolymer is blended or copolymerized with a biodegradable polymer. 30.The method of claim 28 wherein the second polymer is not apolyhydroxyalkanoate.
 31. The method of claim 17 wherein molecules arebound to the polymer, and the molecules are selected from the groupconsisting of molecules which are bioactive, molecules which can bedetected, targeting molecules, and molecules affecting charge,lipophilicity or hydrophilicity of the particle.
 32. The method of claim31 wherein the targeting molecule is selected from the group consistingof compounds specifically reactive with a cell surface component,antibodies and antibody fragments.
 33. The method of claim 17 whereinthe polymer is modified to decrease uptake by the reticuloendothelialsystem.
 34. The method of claim 17 wherein the device is for tissueengineering further comprising seeding cells onto or within the device.35. The method of claim 17 further comprising implanting the device intoa human or animal.
 36. The method of claim 35 wherein the device isimplanted on tissue to form a skin equivalent.
 37. The method of claim35 , wherein the device is formed into a bone prosthesis.
 38. The methodof claim 37 wherein filler materials are mixed with the polymer in anamount effective to enhance the strength of the bone prosthesis.
 39. Themethod of claim 17 further comprising mixing with the polymer structuraland adhesive materials to form a bone cement.
 40. The method of claim 17wherein the polyhydroxyalkanoate has a melting point or glass transitiontemperature less than 136° C.
 41. A biocompatible medical devicecomprising a polyhydroxyalkanoate which does not elicit an acuteinflammatory response when implanted into an animal.
 42. The device ofclaim 41 wherein the device is in a form selected from the groupconsisting of stents, coatings on prosthetic devices, sutures, staples,and tubing.
 43. The device of claim 41 wherein the device is in a formselected from the group consisting of tissue regeneration devices, cellculture devices, vascular grafts, wound dressings, and cell or tissuecoatings.
 44. The device of claim 41 wherein the device is in a formselected from the group consisting of drug, diagnostic or prophylacticdelivery devices.
 45. The device of claim 41 wherein the device is aporous membrane.
 46. The device of claim 41 wherein the device is in theform of microparticles or nanoparticles.
 47. The device of claim 41wherein the device comprises a material selected from the groupconsisting of therapeutic, prophylactic and diagnostic agents.
 48. Thedevice of claim 47 wherein the device comprises a therapeutic agentselected from the group consisting of peptides and proteins, nucleicacids, saccharides and polysaccharides, lipids, synthetic drugmolecules, and imaging agents.
 49. The device claim 41 wherein thedevice is formulated for administration to a mucosal surface.
 50. Thedevice of claim 41 wherein the polyhydroxyalkanoate is a polymer blendor copolymer.
 51. The device of claim 41 wherein molecules are bound tothe polymer, and the molecules are selected from the group consisting ofmolecules which are bioactive, molecules which can be detected,targeting molecules, and molecules affecting charge, lipophilicity orhydrophilicity of the particle.
 52. The device of claim 41 wherein thedevice is for tissue engineering further comprising cells seeded onto orwithin the device.
 53. The device of claim 41 wherein the device isformed into a bone prosthesis.
 54. The device of claim 41 furthercomprising structural and adhesive materials to form a bone cement. 55.The device of claim 41 in the form of a heart prosthetic selected fromthe group consisting of heart valves, heart leaflets, and heart annulus.56. The device of claim 41 in the form of a periodontal graft.
 57. Thedevice of claim 41 in the form of a pericardial patch.
 58. Ahypoallergenic polyhydroxyalkanoate latex.
 59. The latex of claim 58 inthe form of a medical coating or device.
 60. The latex of claim 58 inthe form of surgical gloves.
 61. The latex of claim 58 wherein thepolymer has a melting point of less than 136° C.